DESCRIPTION: The long term goal of our research is to develop effective inhibitors for inward-rectifier K+ channels and use them as molecular probes to explore the molecular mechanisms underlying the function of these ion channels. We have recently identified tertiapin, a honeybee toxin, as a nanomolar-affinity protein-inhibitor for some inward-rectifier K+ channels. Tertiapin, whose structure is known, inhibits the channels by binding to the external vestibule of the K+ conduction pore. In Aim 1A, we will first use tertiapin as a probe to explore the molecular architecture of the outer pore in inward-rectifier K+ channels. By identifying the pairing relation between channel and toxin residues with thermodynamic mutant cycle analysis, we will be able to assign each channel residue pairing with a toxin residue to a location in space with respect to the toxin structure, and thus to delineate the architecture of the outer pore. We will then in Aim 1B examine the mechanism underlying the specificity of channel-toxin interactions. With this knowledge we will design inhibitors with higher channel specificity (Aim 2A), and will also employ phage display, a type of very powerful combinatorial peptide technology, to select specific channel-inhibiting peptides from random peptide libraries (Aim 2B). The proposed fundamental studies also have important medical implications. Inward-rectifier K+ channels are significant both physiologically and pathophysiologically and represent important pharmacological targets. The inhibitors that we develop will be powerful tools both for investigating the physiology and/or pathophysiology of the channels and for developing drugs that specifically target these channels.